Journal: ACS Infectious Diseases

Article Title: Microfluidic Antibody Affinity Profiling Reveals the Role of Memory Reactivation and Cross-Reactivity in the Defense Against SARS-CoV-2

doi: 10.1021/acsinfecdis.1c00486

Figure Lengend Snippet: Microfluidic antibody affinity profiling (MAAP) of nine convalescent COVID-19 patients and three pre-pandemic sera. (A) Probability density plots of MAAP against fluorescently labeled SARS-CoV-2 RBD, spike S1, and spike S2. The graphs show the affinity ( K D ) and the molar concentration of antibody binding sites for each convalescent serum sample. No binding could be detected in the three pre-pandemic sera. Points correspond to the maximum a posteriori values in the two-dimensional posterior probability distribution, and shaded regions correspond to the probability density. Gray shaded regions indicate the area of nonbinding for samples with [antibody binding sites] < K D . (B) ELISA −log (EC 50 ) values for the same nine convalescent COVID-19 patient samples and three pre-pandemic sera ( 1 : NL63+, 229E+; 2 : NL63+, 229E+, OC43+, HKU1+; 3 : 229E+). A SARS-CoV-2 seronegative sample and a SARS-CoV-2 seropositive sample served as negative and positive controls, respectively. Immobilized antigens are SARS-CoV-2 spike ectodomain, spike S1, and the RBD. Detection was performed with fluorescently labeled anti-human IgG antibodies. (A, B) Red boxes/circles indicate convalescent sera 4 and 5 , which have the strongest immune response to all SARS-CoV-2 spike subunits.

Article Snippet: HCoV-NL63 Spike RBD (10605-CV) and HCoV-HKU1 Spike RBD (10600-CV) were obtained from R&D Systems.

Techniques: Labeling, Concentration Assay, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: ACS Infectious Diseases

Article Title: Microfluidic Antibody Affinity Profiling Reveals the Role of Memory Reactivation and Cross-Reactivity in the Defense Against SARS-CoV-2

doi: 10.1021/acsinfecdis.1c00486

Figure Lengend Snippet: Microfluidic antibody affinity profiling against HCoV-NL63 spike S1 and RBD to establish cross-reactivity of antibodies in convalescent COVID-19 serum. (A, B) Equilibrium binding curves of a neutralizing SARS-CoV-2 antibody against 10 nM fluorescently labeled spike S1 from (A) HCoV-HKU1 or (B) HCoV-NL63, respectively. The hydrodynamic radii ( R h ) of the free labeled spike proteins did not increase upon addition of the antibody, indicating the absence of binding. (C) Equilibrium binding curve of an anti-HKU1 antibody against 10 nM fluorescently labeled spike S1 of HCoV-HKU1 showed very tight binding with a K D below 0.1 nM. The K D was determined by nonlinear least-squares fitting using eq . (D) Probability density plots of MAAP against fluorescently labeled HCoV-NL63 spike S1 and HCoV-NL63 RBD. The graphs show the affinity ( K D ) and the molar concentration of antibody binding sites for each of the convalescent COVID-19 (red) and the pre-pandemic sera (blue), whereby pre-pandemic sera and 2 were found to be seropositive for HCoV-NL63. Points correspond to the maximum a posteriori values in the two-dimensional posterior probability distribution, and shaded regions correspond to the probability density.

Article Snippet: HCoV-NL63 Spike RBD (10605-CV) and HCoV-HKU1 Spike RBD (10600-CV) were obtained from R&D Systems.

Techniques: Binding Assay, Labeling, Concentration Assay

Journal: ACS Infectious Diseases

Article Title: Microfluidic Antibody Affinity Profiling Reveals the Role of Memory Reactivation and Cross-Reactivity in the Defense Against SARS-CoV-2

doi: 10.1021/acsinfecdis.1c00486

Figure Lengend Snippet: (A) Schematic of SARS-CoV-2–HCoV-NL63 cross-reactivity competition assay. Typically, 10 nM fluorescently labeled HCoV-NL63 RBD was mixed either with buffer or with patient serum, incubated for 1 h, and subjected to microfluidic diffusional sizing to determine the size of the free labeled RBD ( R h,free ) and the size of the immune-complex ( R h,complex ). For binding competition, 250 nM unlabeled SARS-CoV-2 RBD was added to the mixture of 10 nM HCoV-NL63 RBD and patient serum and incubated for 1 h before measuring the hydrodynamic radius ( R h ). If the serum contains cross-reactive antibodies, a size decrease is observed, as the antibodies will bind to the excess of unlabeled RBD (left box). If the antibodies in the serum are not cross-reactive, they remain bound to the labeled HCoV-NL63 RBD, and the R h of the immuno-complex will stay constant (right box). (B) SARS-CoV-2–HCoV-NL63 cross-reactivity competition assay in convalescent COVID-19 sera and pre-pandemic sera. Filled circles (●) indicate the size of the HCoV-NL63 RBD–antibody immune complex. Empty circles (○) indicate the size of HCoV-NL63 RBD after addition of excess unlabeled SARS-CoV-2 RBD. (Red) Convalescent COVID-19 sera, (Blue) pre-pandemic sera. No decrease of R h upon addition of the unlabeled SARS-CoV-2 RBD could be observed in any of the sera, indicating no cross-reactivity of anti-NL63 antibodies with SARS-CoV-2 RBD.

Article Snippet: HCoV-NL63 Spike RBD (10605-CV) and HCoV-HKU1 Spike RBD (10600-CV) were obtained from R&D Systems.

Techniques: Competitive Binding Assay, Labeling, Incubation, Binding Assay